Protein molecular weight marker

SDS-PAGE was performed according to the method described by Schägger and Von Jagow [28 (link)] using 4% stacking gel (w/v) and 12% polyacrylamide gel (w/v). First, 10 milligrams of protein isolate was dissolved in 1 mL of denaturing buffer for samples, (0.5 M Tris-HCl pH 6.8, glycerol, 10% SDS, 0.5% bromophenol blue, β-mercaptoethanol) and heated at 95 °C. Then, 10 μL of the sample was loaded into the sample wells. Protein separation was performed at 80 V for 30 min, then at 110 V for 90 min for a separation gel using a Mini Protean II device (Bio-Rad Laboratories, Hercules, CA, USA). The gel was stained with brilliant blue for 40 min (Bio-Rad Coomassie R250, Bio-Rad Laboratories, Hercules, CA, USA). Gel bleaching was performed three times using water/methanol/acetic acid (7/2/1 v/v/v) for 15 min each shaking cycle using an orbital shaker (Fristek S10, Taichung, Taiwan). The molecular weight of proteins was evaluated using a protein molecular weight marker (250 to 10 kDa, Bio-Rad Laboratories, Hercules, CA, USA) loaded at a dose of 5 μL into the sample well. Gels were scanned with an E-Box VX5 (Vilber Lourmat, Paris, France), and captured image analysis was performed using Vision Capt software (V16.08a, Vilber Lourmat, Paris, France).

Serum MMP-2 activity was determined by zymography according to Storch et al. (25 (link)). Briefly, serum samples were subjected to electrophoresis in a 12% polyacrylamide gel and copolymerized with 1% gelatin. An internal standard (control serum sample) and a protein molecular weight marker (Bio-Rad, USA) were used to analyze and compare the gels. Gels were stained with 5% Coomassie blue R-250 Brilliant Blue (Sigma-Aldrich) and destained with a solution of 50% methyl alcohol and 10% glacial acetic acid. Scion Image software (Scion Corporation, USA) was used by a blinded researcher to quantify band intensity; the more intense the band, the higher the enzyme activity. Inactive (pro-MMP-2) and active forms of MMP-2 were identified as bands at 72 and 64 kDa, respectively. Figure 1 shows a representative image of the zymography gel.

10% acrylamide gels were run at 100 volts for 100 minutes to resolve the purified samples along with a protein molecular weight marker (Bio-Rad, USA) and used for coomasive brilliant blue R-250 staining. Resolved proteins in SDS-PAGE gels were transferred to a nitrocellulose membrane and non-specific sites were blocked with 5% milk. In-house monoclonal antibodies against VP1 (mAb 51) [23 (link)], VP2 (mAb 7C7) [24 (link)] and polyclonal antibody against VP3 (raised against rVP3 from E.coli) were used as primary antibodies. Goat anti-mouse antibody (in 5% skim milk), conjugated with HRP, was used as secondary antibody. Detection was done using Enhanced Chemiluminescence (ECL) Plus western blotting detection reagent (GE Healthcare, UK).

Proteins from Der p. and B. tropicalis extracts were separated by electrophoresis with use of reduced conditions and 15% polyacrylamide running gel according to Laemmli [22 (link)]. All samples of each commercial allergenic extract were loaded with equal protein content (0,5 µg/lane). A protein molecular weight marker (Bio-Rad, Hercules, CA, USA) was used as a standard. Proteins were visualized using Silver dye staining (Thermo Fisher Scientific, Waltham, USA).